Transfection kinetics and structural promoters

ABSTRACT

The invention features methods of analyzing the kinetics properties of transfection reactions. Also featured are methods for creating structural promoters which are effectively unregulated by enhancers and repressors. The structural promoters are significantly more active than the native promoter sequences upon which they are based.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part and claims the benefit of thefiling date of U.S. provisional application 60/464,434, filed Apr. 22,2003, PCT application US02/33669, filed Oct. 22, 2002, and U.S.provisional application 60/342,788, filed Oct. 22, 2001.

BACKGROUND OF THE INVENTION

Improved methods are needed for measuring the kinetics of transfectionof nucleic acid into cells and for measuring promoter activity of thenucleic acid once it is in the cell. Additionally, it would be desirableto have methods for more efficiently expressing RNA or protein moleculesof interest within a target cell.

SUMMARY OF THE INVENTION

This invention provides methods for quantifying and describing thekinetics of transfection, a process by which expression of RNA orprotein is observed following introduction of DNA into cells. Alsoprovided are permanent and transient forced-open complexes insupercoiled DNA or RNA, as well as methods for creating such complexes.These complexes function as strong transcriptional promoters.

In one aspect, the effect of any expression element on transfection ismeasured by (i) transfecting a host cell with several (e.g., at least 2,3, 4, 5, 8, 10, 15, 20, 30, or more) sub-saturating concentrations of avector comprising the expression element; (ii) measuring the activity ofa reporter protein; (iii) measuring the expression of the same reporterprotein under the same transfection conditions transfected into controlhost cells using a vector that is deficient in the expression elementunder study; and (iv) comparing the expression levels using an inverseplot transformation.

In another aspect, the invention provides a method of expressing an RNAor protein that includes the steps of (i) annealing to a supercoiled ordouble stranded DNA or RNA an oligonucleotide that is complementary to asequence upstream from the RNA or protein coding sequence of interest ofthe supercoiled or double stranded DNA or RNA and (ii) introducing theresulting annealed product into a host cell under conditions that resultin expression of the RNA or protein by the host cell. Desirably, theoligonucleotide is annealed to the non-template strand of DNA or RNA. Invarious embodiments, the host cell may be in cell culture, in a tissue,or in an organism.

In desirable embodiments, the supercoiled DNA or RNA is an expressionconstruct or expression vector. Desirable expression constructs containa transcriptional promoter operably linked an RNA or protein codingsequence of interest. Desirably, the oligonucleotide forms aheteroduplex with the supercoiled DNA or RNA. In another desirableembodiment, the oligonucleotide is complementary to a naturallyoccurring or artificially inserted promoter sequence. Theoligonucleotide comprises at least 10 nucleotides, desirably at least 20nucleotides, more desirably at least 30 nucleotides, most desirably atleast 40 nucleotides, or even at least 50 nucleotides complementary tothe DNA.

In another aspect, the invention provides a composition that includes aDNA expression construct or expression vector, desirably supercoiled,having a torsionally locked single stranded oligonucleotide or padlockoligonucleotide annealed to the DNA upstream from an RNA or proteincoding sequence of interest. Desirably, the oligonucleotide forms aheteroduplex with the supercoiled DNA or RNA. In another desirableembodiment, the oligonucleotide is complementary to a naturallyoccurring or artificially inserted promoter sequence. In variousembodiments, the oligonucleotide is a DNA, an RNA, a PNA, or a mixturethereof.

In desirable embodiments of any of the aspects of the invention, thesingle stranded oligonucleotide comprises at least five contiguousnucleotides at the 5′ terminus that are complementary to a first regionof the supercoiled DNA or RNA and at least five contiguous nucleotidesat the 3′ terminus that are complementary to a second region of thesupercoiled DNA or RNA that is adjacent to the first region. Desirably,the first and/or second regions of the supercoiled DNA or RNA that areat least 80, 85, 90, 95, or 100% complementary to the 5′ or 3′ terminusof the single stranded oligonucleotide include at least 10 to 20nucleotides, more desirably at least 30 nucleotides, most desirably atleast 40 nucleotides, or even at least 50 nucleotides or 100nucleotides. Desirably, at least 5 contiguous nucleotides, moredesirably at least 6 contiguous nucleotides at each of the 5′ and the 3′termini of the single stranded oligonucleotide are 100% complementary tothe corresponding regions of the supercoiled DNA or RNA. In addition tothe complementary 5′ and 3′ sequences, the oligonucleotide desirably hasa region linking the 5′ and the 3′ sequences that is at least as long,and desirably longer, than the total length of the complementary 5′ and3′ sequences. Desirably, the linking region consists of nucleotides.

In another aspect, the invention provides a method of expressing an RNAor protein. This method includes (i) providing a single strandedoligonucleotide comprising nucleotides at its 5′ terminus (e.g., thefirst five nucleotides at the 5′ terminus) that are complementary to afirst region of a supercoiled DNA or RNA expression vector or expressionconstruct and nucleotides at its 3′ terminus terminus (e.g., the lastfive nucleotides at the 3′ terminus) that are complementary to a secondregion of the supercoiled DNA or RNA that is adjacent to the firstregion; (ii) annealing the oligonucleotide to the supercoiled DNA or RNAsuch that the 5′ and the 3′ termini of the oligonucleotide arejuxtaposed and base-pair with the first and second regions,respectively, of the supercoiled DNA or RNA; (iii) ligating the 3′terminus of the oligonucleotide to the 5′ terminus to form a circularoligonucleotide that is topologically linked to the supercoiled DNA orRNA; and (iv) introducing the resulting DNA or RNA with thetopologically linked oligonucleotide into a cell under conditions thatallow expression of an RNA or protein encoded by the supercoiled DNA orRNA. The cell may optionally be in cell culture, in a tissue, or in anorganism.

In another aspect, the invention features a chimeric HCMV/EF-1 α Pminpromoter. In a related aspect, the invention provides a nucleic acid(e.g., DNA or RNA) expression construct that includes a chimericHCMV/EF-1 αPmin promoter operably linked to an RNA or protein codingsequence of interest.

In another aspect, the invention features a composition that includes anHCMV promoter annealed to a torsionally locked oligonucleotide.Desirably, the promoter is operably linked to an RNA or protein codingsequence of interest.

In desirable embodiments of any of the aspects of the invention, the 3′and 5′ termini of the single stranded oligonucleotide are each 100%complementary to at least five contiguous nucleotides of the supercoiledDNA or RNA or at least 80, 85, 90, 95, or 100% complementary to at least6, 10, 15, 20, or 30 (in order of increasing preference) contiguousnucleotides of the supercoiled DNA or RNA. In desirable embodiments, thecomplementary regions of the supercoiled DNA or RNA are upstream from anRNA or protein coding sequence of interest. Desirably, the regions ofcomplementarity in the supercoiled DNA or RNA are contiguous and arecontained within a promoter region (e.g., a TATA and/or CAT boxpreceding the transcription initiation site of a coding sequence oninterest, at least 45 nucleotides prior to the initiating ATG codon ofthe coding sequence, a region beginning at position −1 relative to thetranscription initiation site of the coding sequence, or a region atpositions −10 to −70 relative to the transcription initiation site ofthe coding sequence). The coding sequence of interest in the supercoiledDNA or RNA (e.g., circular, nicked, or linear DNA or RNA) may or may notbe operably linked to a promoter. Desirably, the oligonucleotidemodulates the expression of the RNA or protein of interest (e.g.,increases or decreases expression by at least 2, 3, 5, 10, 15, 20, 30,or 40-fold). In some embodiments, the oligonucleotide has an internalregion (e.g., a region linking the 3′ and 5′ termini that is notbase-paired with the supercoiled DNA or RNA) that is less than 50, 40,30, 20, 10, or 5% complementary to the supercoiled DNA or RNA.

In other embodiments, the oligonucleotide includes one or more modifiednucleotides in which the 2′ position in the sugar contains a halogen(such as flourine group) or contains an alkoxy group (such as a methoxygroup) which increases the half-life of the oligonucleotide in vitro orin vivo compared to the corresponding oligonucleotide in which thecorresponding 2′ position contains a hydrogen or an hydroxyl group. Inyet other embodiments, the oligonucleotide includes one or more linkagesbetween adjacent nucleotides other than a naturally-occurringphosphodiester linkage. Examples of such linkages include phosphoramide,phosphorothioate, and phosphorodithioate linkages. In other embodiments,the oligonucleotide contains sequence or structure that binds regulatoryfactors, for example, a regulatory sequence (e.g., a transcriptionfactor binding site, a promoter, or enhancer), or the oligonucleotidemay contain non-nucleotide entities (e.g., for receptor binding,intracellular targeting, or endosomal disruption).

By “expression element” is meant any feature or sequence of a DNAmolecule that affects transcription or translation of a nucleic acidsequence. Examples of expression elements include promoters, enhancers,repressors, polyadenylation sites, and introns. Expression elements thatcan be assessed using this invention also include protein elements suchas transcriptional or translational enzymes, for example, polymerasesand transcription factors.

By “expression vector” is meant any double stranded DNA or doublestranded RNA designed to transcribe an RNA, e.g., a construct thatcontains at least one promoter operably linked to a downstream gene orcoding region of interest (e.g., a cDNA or genomic DNA fragment thatencodes a protein, or any RNA of interest, optionally, e.g., operativelylinked to sequence lying outside a coding region, an antisense RNAcoding region, a dsRNA coding region, or RNA sequences lying outside acoding region). Transfection or transformation of the expression vectorinto a recipient cell allows the cell to express RNA or protein encodedby the expression vector. An expression vector may be a geneticallyengineered plasmid, virus, or artificial chromosome derived from, forexample, a bacteriophage, adenovirus, retrovirus, poxvirus, orherpesvirus.

By an “expression construct” is meant any double-stranded DNA ordouble-stranded RNA designed to transcribe an RNA, e.g., a constructthat contains at least one promoter operably linked to a downstream geneor coding region of interest (e.g., a cDNA or genomic DNA fragment thatencodes a protein, or any RNA of interest). Transfection ortransformation of the expression construct into a recipient cell allowsthe cell to express RNA or protein encoded by the expression construct.An expression construct may be a genetically engineered plasmid, virus,or artificial chromosome derived from, for example, a bacteriophage,adenovirus, retrovirus, poxvirus, or herpesvirus. An expressionconstruct does not have to be replicable in a living cell, but may bemade synthetically.

By “commitment to expression” is meant the likelihood of transcriptionalinitiation. The commitment to expression is quantified numerically bythe K_(m). Commitment to expression is affected by the affinity of thepolymerase for the promoter and by the probability of all of the stepsthat precede initiation of transcription (i.e., the steps prior to theformation of the first phosphodiester linkage).

By “torsionally locked oligonucleotide” or “padlock oligonucleotide” ismeant is a circular nucleic acid (e.g., DNA, RNA, DNA/RNA hybrid, orPNA) or peptide nucleic acid that goes in and out between the twostrands of a double-stranded DNA or RNA helix (e.g., circular, linear,or nicked supercoiled DNA or RNA). Padlocks are illustrated in FIG. 12and described further by Escude et al. (Proc Natl Acad Sci U S A1999;96(19):10603-7) and Nilsson et al. (Science.1994;265(5181):2085-8), which are both hereby incorporated by reference.

By “non-padlocked oligonucleotide” is meant a single-stranded linear orcircular nucleic acid (e.g., DNA, RNA, PNA, or hybrids thereof) annealedas described herein to one strand of a double stranded DNA or RNA helix,but not torsionally locked between the two strands of such DNA or RNA.

By “operably linked” is meant that a nucleic acid molecule and one ormore regulatory sequences (e.g., a promoter) are connected in such a wayas to permit transcription of the mRNA or permit expression and/orsecretion of the product (i.e., a polypeptide) of the nucleic acidmolecule when the appropriate molecules are bound to the regulatorysequences.

By a “promoter” is meant a nucleic acid sequence sufficient to directtranscription of a covalently linked nucleic acid molecule. Alsoincluded in this definition are those transcription control elements(e.g., enhancers) that are sufficient to render promoter-dependent geneexpression controllable in a cell type-specific, tissue-specific, ortemporal-specific manner, or that are inducible by external signals oragents; such elements, which are well-known to skilled artisans, may befound in a 5′ or 3′ region of a gene or within an intron. Desirably apromoter is operably linked to a nucleic acid sequence, for example, acDNA or a gene in such a way as to permit expression of the nucleic acidsequence.

By “reporter gene” is meant any gene that encodes a product whoseexpression is detectable and/or able to be quantitated by immunological,chemical, biochemical, or biological assays. A reporter gene productmay, for example, have one of the following attributes, withoutrestriction: fluorescence (e.g., green fluorescent protein), enzymaticactivity (e.g., β-galactosidase, luciferase, chloramphenicolacetyltransferase), toxicity (e.g., ricin A), or an ability to bespecifically bound by an additional molecule (e.g., an unlabeledantibody, followed by a labelled secondary antibody, or biotin, or adetectably labelled antibody). It is understood that any engineeredvariants of reporter genes that are readily available to one skilled inthe art, are also included, without restriction, in the foregoingdefinition.

By “transformation” or “transfection” is meant any method forintroducing foreign molecules into a cell (e.g., a bacterial, yeast,fungal, plant, insect, or animal cell, particularly a vertebrate ormammalian cell). The cell may be in an animal. Lipofection,DEAE-dextran-mediated transfection, microinjection, protoplast fusion,calcium phosphate precipitation, viral or retroviral delivery,electroporation, and biolistic transformation are just a few of thetransformation/transfection methods known to those skilled in the art.The nucleic acid may be, for example, naked RNA or DNA or a localanesthetic complexed to RNA or DNA. Other standardtransformation/transfection methods and other RNA and/or DNA deliveryagents (e.g., a cationic lipid, liposome, or bupivacaine) are describedin WO 00/63364, filed Apr. 19, 2000 (see, for example, pages 18-26).Commercially available kits can also be used to deliver RNA or DNA to acell (e.g., Transmessenger Kit from Qiagen, an RNA kit from XeragonInc., and an RNA kit from DNA Engine Inc. (Seattle, Wash.)).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of reporter gene expression as a function of theplasmid DNA concentration used in the transfection procedure. Theconcentration of reporter plasmid DNA expressing beta-galactosidase(β-gal) or secreted alkaline phosphatase (SEAP) was varied as indicatedon the X-axis, from 50 ng to 2.5 micrograms. The total DNA concentrationwas kept constant at 2.5 micrograms by adding a promoterless luciferaseplasmid as filler DNA. The DNA was then complexed with Lipofectamine™ toensure that similar Lipofectamine complexes were applied to the cells,and these complexes were added to RD cells. The transfection process wasas described in Example 5C. The cells were lysed to determine β-galactivity or culture supernatants were used to measure SEAP activity.Cells were lysed as described herein. β-gal activity was measured asdescribed in Molecular Cloning, A Laboratory Manual, 2 nd Edition.Sambrook, J., Fritsch, E. F. and Maniatis, T.; Cold Spring Harbor Press,Plainview, N.Y. (1989); however, the assay was carried out kineticallyas described herein. The initial velocities obtained as described hereinwere plotted as a function of varying amounts of reporter DNA in thetransfection mixes.

FIGS. 2A and 2B are a series of graphs showing the β-gal enzymaticactivity and mRNA levels in RD cells following transfection with varyingamounts of vector DNA. The mRNA was quantified using a Phosphorimager(Molecular Dynamics, Sunnyvale, Calif.) of Northern blots hybridizedusing a radioactive β-gal or SEAP specific DNA probe. Northern blottingwas performed according to the methods described in Sambrook et al.,Cold Spring Harbor Press, Plainview, N.Y. (1989). The mRNA was alsoquantified using a quantitative RT-PCR method using oligonucleotidesdesigned to specifically prime and amplify target mRNAs.

FIGS. 3A-3D are a series of graphs showing that the transfection process(transfection) is amenable to steady state kinetic analysis. To enablesteady state kinetic analysis, the amount of product formed has to belinear with respect to the amount of cells in the transfection mixture,linear with respect to time following transfection, and exhibit aMicaelis-Menten substrate (DNA) saturation profile with linearity at DNAconcentrations below the K_(m). Transfections were carried out asdescribed previously. Cell numbers and the amounts of DNA were varied asindicated.

FIGS. 4A and 4B are a series of graphs comparing β-gal activityfollowing transfection under sub-saturating conditions in RD cells.β-gal is under the control of either the HCMV, SCMV, or HCMVm promoter.The plots are of inverse values for initial velocity (Y-axis) and forsubstrate (X-axis) in Lineweaver-Burk format.

FIG. 5 is a graph comparing the β-gal activity in RD cells followingtransfection with vectors containing either an HCMV or SCMV promoter.Single construct transfections are compared to mixed transfections asdescribed herein.

FIGS. 6A and 6B are graphs comparing the effects of inclusion ordeletion of the enhancer element of EF-1α promoter.

FIGS. 7A and 7B are graphs comparing the effects of the HCMV and EF-1αPmin promoters on β-gal expression in RD cells after transfection usingsub-saturating conditions. Saturation of transfection results fromlimiting amounts of one or more of the components or steps in thetransfection machinery (e.g., DNA uptake, nuclear transport of DNA,transcription factors, polymerase, triphosphoribonucleosides, splicing,capping, polyadenylation, transport of mRNA to the cytoplasm, ribosomebinding, translation, protein folding, translocation of the protein intothe ER).

FIG. 8 is a schematic diagram of the chimeric HCMV/EF-1α Pmin promoterdescribed in Example 2. The EF-1α Pmin promoter replaces the 3′ terminal71 base-pairs of the HCMV promoter and is adjacent to coordinate 1070 ofthe HCMV promoter according to GenBank Accession # X03922.

FIGS. 9A and 9B are graphs comparing the effects of the HCMV and RSVpromoters on β-gal expression in RD cells after transfection usingsub-saturating conditions.

FIG. 10 is a schematic of open complex formation under normal(transcription factor/polymerase-induced) and forced conditions.

FIG. 11 is a schematic showing the natural “breathing” of supercoiledDNA, and a strategy for creating an open complex by forming aheteroduplex within the supercoil.

FIG. 12 is a schematic demonstrating the molecular mechanism fortopologically locked heteroduplex formation in a DNA supercoil (padlock)(SEQ ID NOS:11-14.)

FIG. 13 is a schematic showing the strategy used for designingoligonucleotides useful for heteroduplex formation.

FIGS. 14A and 14B are graphs comparing the β-gal activity in RD cellstransfected under sub-saturating conditions in which the vector wasincubated with synthetic oligonucleotides of varying lengths, sequences,and specificities. Both the Michalis-Menten and Lineweaver-Burk plotsare shown.

FIGS. 15A and 15B depict an electrophoretic separation of vector DNAfollowing incubation and enzymatic ligation with a ³²P-labeled “padlock”oligonucleotide. FIG. 15A is a picture of an ethidium bromide stainedgel, and FIG. 15B is a picture of an autoradiogram (Phoshorimager,Molecular Dynamics, Sunnyvale, Calif.) of the same gel. The three lanesin the gels are DNA markers (M), a Betagal plasmid preparation thatcontains linear, open circular, and supercoiled forms (017), and allthree plasmid forms of pShooter (Invitrogen, Carlsbad, Calif.) ascontrol (PS) (a plasmid without betagal sequences). FIG. 15B has aradioactive band associated only with the SC form of the betagal (017)plasmid. No radioactivity is associated with the control pShooterplasmid (PS) or with the marker DNA (M). The padlock sequences weredesigned to base-pair with the betagal sequences, and the padlockingreactions were carried out against mixed plasmid forms of both betagal(017) and pShooter (PS) preparations. The figure demonstratesspecificity to supercoiled DNA and further proves that the supercoiledDNA “breathes,” and that an oligonucleotide can indeed invade andhybridize to the single stranded loops generated.

FIG. 16 depicts padlock oligonucleotide #1 being annealed to pCMV-betaas described herein. The 5′ twenty-eight nucleotides of the padlockoligonucleotide base-pair with nucleotides mapping to coordinates −47 to−20 of the HCMV promoter present in pCMV-beta, and the 3′ twentynucleotides base-pair to nucleotides mapping to coordinates −48 to −67.Therefore, the 3′ and the 5′ ends are base-paired to adjacentnucleotides (“*,” nucleotides −47 and −48) of the HCMV Promoter inpCMV-beta and are positioned for ligation. The internal nucleotideslinking the 3 ′ and 5′ sequences do not base-pair with pCMV-beta. Thesenucleotides (i) give the oligonucleotide freedom to rotate its terminito facilitate base-pairing and (ii) prevent additional torsional stressto the supercoiled pCMV-beta following annealing of the padlock oligo.Once annealed, the oligo is ligated to create a phosphodiester linkagebetween the 3′ and the 5′ ends of the oligo (*) generating the padlockedoligo structure. Regions flanking the annealed padlock oligo aresingle-stranded due to torsional constraints.

FIG. 17 depicts the various EF-1α Promoter constructs described herein.PE is comprised of a promoter element, EF-1α Pmin, and an enhancer. Theenhancer is comprised of an intron and two flanking exons, x and y. Pxis comprised of the promoter element, EF-1α Pmin, and x and y arederived from the enhancer element. Pmin is the minimal promoter element,EF-1α Pmin.

FIG. 18 depicts the desirable features for padlocked and non-padlockedoligonucleotides. The oligo desirably does not base-pair with sequencesdownstream from the −1 position of the promoter of interest and topreferentially base-pair with sequences located between −1 and −45 ofthe promoter of interest. The desirable maximal length of the oligo is100 nucleotides and the desirable minimal length is 12 nucleotides.

FIG. 19 depicts the method for designing padlocked and non-padlockedoligonucleotides in situations in which there is not a promoter or thelocation of the promoter is unknown. For transcription ofprotein-encoding RNAs, oligonucleotides are designed to base-pair atleast 20 nucleotides upstream from an initiation codon, more desirably21-50 nucleotides upstream from the initiation codon, and most desirably51-80 nucleotides upstream from the initation codon. For transcriptionof RNA sequences that are not translated, oligonucleotides are desirablydesigned to anneal to sequences located about 1-45 nucleotides upstreamof the RNA sequences to be transcribed. Desirably, the maximal length ofthe oligo is 100 nucleotides, and the desirable minimal length is 12nucleotides.

FIG. 20 depicts the positions of three DNA oligonucleotides (each46-mers; SC1001, SC1002, and SC1003) relative to their complementaryregions of the HCMV promoter.

DETAILED DESCRIPTION

Transfection of DNA into cells is the primary step in the analysis ofgene expression. Quantitative analysis of gene expression requires thattransfection efficiency differences do not compromise the comparativeanalysis of expression elements. The present invention relates tomethods for analyzing the relative expression of transfectionconstructs. The methods are based on our discovery that transfection isa saturable process, and that the expression profile of a reporterconstruct is amenable to steady state kinetic analysis.

In a one aspect of the invention, using the analytical methods, we havere-defined gene promoters by structural, rather than sequence, criteria.Based on the discovery of the structural characteristics of promoters,we provide a strategy for creating super promoters that aresignificantly stronger than the native promoters on which they arebased. Additionally, this strategy can be used to create a functionalpromoter site at genetic sites that do not encode any expressionelements.

Another aspect of the invention provides improved artificial promoterscreated by linking sequences demonstrated through the analytical methodsof the invention to have desirable kinetic characteristics.

In another aspect, the invention provides DNA expression constructs andexpression vectors, desirably supercoiled, having a torsionally lockedsingle stranded oligonucleotide or padlock oligonucleotide annealed tothe DNA upstream from an RNA or protein coding sequence of interest.Also provided are DNA expression constructs and expression vectors,desirably supercoiled, having a single stranded oligonucleotide annealedto a DNA sequence upstream from the protein or RNA coding sequence ofinterest. In desirable embodiments, the single stranded oligonucleotideis annealed to a naturally occurring or artificially inserted promotersequence.

Transfection Kinetics

The expression of various genetic elements can be described using thestandard kinetic constants, V_(max) and K_(m). The K_(m) is a measure ofthe commitment to expression of a genetic element. The lower the K_(m),the greater the commitment. The K_(m) therefore defines the amount ofDNA expression construct required to be effective. Low K_(m) indicatesthat the expression machinery can sense even smaller quantities ofinternalized DNA as compared to the amounts of sequences that havehigher K_(m). Delivery of DNA expression vectors is inefficient in vivo.Therefore, it is desirable to include elements of a promoter that has anexceedingly low K_(m). The V_(max) is a measure of the relativeexpression potential of the element at infinite (unlimited)concentrations of template DNA (expression vector). K_(m) and V_(max) donot co-segregate with the same DNA sequences, and are predicted to beassociated with definable DNA sequences. Therefore, it is possible tocreate artificial promoters by using elements of low K_(m) and highV_(max). In some embodiments, a promoter is created by combining aregion associated with a low K_(m) with a region associated with a highV_(max).

Cell culture transfection typically results in the cellular uptake ofhundreds of DNA molecules per cell, and is a process that can besaturated. At transfection saturation, protein expression levels plateausuch that further increases of transfecting DNA do not result inproportional increases in transgene expression. At saturation, at leastone of the components of the transfection process is limiting.Accordingly, meaningful and accurate measurements of relative expressionlevels must be taken at sub-saturating concentrations. Thesub-saturating concentrations are often orders of magnitude smaller thanthose used for in vitro transfection, and may be more applicable for invivo applications.

We have invented a matrix transfection assay that measures proteinexpression in the linear range of transfection, as a true measure of thedifferences among expression elements. The assay also allowsdiscrimination of transfection efficiency effects. Using this assay, wehave discovered that: (1) transfection is saturable; (2) saturation oftransfection occurs at a post-transcriptional step; and (3)co-transfection is not a reliable indicator of transfection efficiencywhen performed under saturating conditions.

EXAMPLE 1 Measuring the Kinetics of Transfection

Transfections were performed using cationic lipid (Lipofectamine)complexed DNA in human Rhabdomyosarcoma (RD) cells. Amounts oftransfected reporter gene plasmid in transfection mixes ranged from 50ng to 2.5 μg/transfection. The total amount of DNA per transfection washeld constant at 2.5 μg by adding a promoterless control plasmid to eachtransfection reaction.

A variety of reporter plasmids were used. Plasmids were designed toexpress β-galactosidase or human secreted alkaline phosphatase (SEAP),and contained various promoter elements including the HCMV, SCMV (Changet al., J. of Virology, 64: pp 264-277, 1990;) HCMVm (e.g., a HCMVpromoter with a gfi transcription factor binding sequence deleted),Zweidler-McKay et al., Molecular and Cellular Biology, 16: pp 4024-4034,1996), and elongation factor 1 alpha (EF-1α) minimal and completepromoters (WO 02/50264A2). Additionally, intron-containing andintronless vectors were compared.

Enzyme Expression Analysis

Beta-galactosidase (βgal) activity was measured in the lysates oftransfected cells using a calorimetric kinetic enzyme assay, andnormalized to total cellular protein. SEAP activity was measured in themedia of transfected cells. Expression is plotted as the initialvelocity of the reaction for each DNA concentration tested. Enzymaticactivity was measured at several time points during the reaction. Themeasurements of the reaction over time were plotted. Slope of the linearportion of the reaction curve is the initial velocity rate utilized inthe transfection plots. The initial velocity of the enzymatic reactionsis directly proportional to the amount of the enzyme present. Enzymaticassays were performed at 1, 2, and 4 days post-transfection. Allvectors, regardless of cell type, resulted in transfection saturation(FIG. 1). For different combinations of vector and cells, saturationoccurred at different amounts of DNA in the transfection mixture.Saturation occurred at expression vector levels ranging from 200 ng-1 μgper 6-7×10⁵ cells.

RNA Analysis

Total RNA was isolated from cells using the StrataPrep Total RNAMiniprep Kit (Stratagene). Three micrograms of total RNA was run on a1.2% agarose-formaldehyde gel, transferred to Zeta-probe membrane(Bio-Rad), and probed with β-gal sequences. After initial hybridization,the membrane was stripped and probed for actin RNA. Probes were³²P-labeled using random primed synthesis. The resulting signals werevisualized and quantitated by phosphorimager analysis. Relative RNAvalues were obtained by normalizing β-gal RNA to actin RNA.

Experiment #1

FIG. 2 shows representative results of β-gal enzymatic activity and mRNAlevel, as a function of vector DNA concentration. Experiments wereperformed in duplicate. β-gal expression was found to be linear up to 1μg of transfected reporter plasmid, after which, expression levelsbecame saturated. β-gal RNA levels were linear over the entire range ofDNA concentration used. Replicates exhibited less than 5% variation.Together, these data show that saturation of transfection (proteinexpression) occurs at a post-transcriptional step.

Sub-saturating concentrations of β-gal vector DNA were used toinvestigate other kinetic properties of the transcription system. FIG. 3shows that β-gal activity increases linearly, under sub-saturatingconditions, with increasing time after transfection (A and B), with celldensity (C).

Experiment #2

Sub-saturating amounts of β-gal reporter plasmid (200 ng) wereco-transfected with a super-saturating amount of a second plasmid (2.3μg). The second plasmid was either HCMV-HSVgD (expresses a proteinproduct), HCMV-HPVL1 (expresses mRNA that is transcribed, transported tothe cytoplasm, but not translated), or pLUC (promoterless plasmid thatis not transcribed). The HCMV sequence has Genbank Accession number X03922.

β-gal protein and mRNA were measured and normalized as previouslydescribed. The lowest expression of protein and RNA was assigned anarbitrary value of one, and the ratio of β-gal protein:RNA was compared.

Approximately equal amounts of mRNA are made by the HCMV-HSVgD and theHCMV-HPVL1 plasmids, as measured by RT-PCR. There was no difference inthe amount of β-gal protein per unit β-gal RNA in cells co-transfectedwith non-protein coding plasmids (HCMV-HPVL1) compared to cellstransfected without a competing plasmid (HCMV-HSVgD). However, in cellsco-transfected with plasmids that directed the synthesis of a protein(HCMV-HSVgD), 14-18 fold less β-gal protein per unit β-gal RNA wasproduced (compare pLUC or HCMV-HPVL1 to HCMV-HSVgD). RNA levelsproportionately increase with DNA transfected, while protein levels donot. Moreover, the expression of protein is competed with higherconcentrations of only protein-expressing plasmids (as opposed toplasmids that express mRNA which is not translated). This resultindicates that the limiting elements of expression involvepost-transcriptional steps and that saturation occurspost-transcriptionally.

TABLE 1 Competition Experiment to Define Saturation Expression β-galprotein β-gal β-gal Construct Block level RNA level protein:RNAHCMV-HSVgD no block 1 1.8 0.56 HCMV-HPVL1 translation 8-10 1 8-10 pLUGtranscription 8-10 1 8-10Experiment #3

Kinetic constants of two different promoter constructs were compared.FIG. 4 is an inverse plot of initial velocities as a function of DNAconcentration (Lineweaver-Burk plot). The V_(max) is derived from theY-axis intercept, and the K_(m) from the X-axis intercept. The HCMVpromoter, when compared with the SCMV promoter (Chang et al., J. ofVirology, 64: pp 264-277, 1990) demonstrates differences in initialvelocity rates. Therefore, even at saturating substrate concentrations,these two promoters are predicted to have different levels of activity(HCMV>SCMV). The similarity of K_(m) values suggests similar commitmentsto catalysis.

When the HCMV promoter is compared with the HCMVm version harboring amutation in the gƒi box (transcriptional repressor site) (Zweidler-McKayet al., Molecular and Cellular Biology, 16: pp 4024-4034, 1996) the twopromoters have significant differences in their initial velocity rates,but at saturating concentrations of DNA, they are predicted to havesimilar velocities (V_(max)). The promoters do, however, have differentK_(m) values, with the mutant showing a higher commitment to catalysis(lower K_(m)). K_(m) differences reflect differences in events that leadto transcription initiation resulting from differences in affinity forthe transcription machinery. V_(max) differences reflect, in mostinstances, the activity following transfection with infinite DNAconcentrations.

Experiment #4

The relative activity of the HCMV promoter and the SCMV promoter in RDcells was compared (FIG. 5). Transfection was performed in the linearrange of DNA concentration, and enzyme activity results were correctedfrom transfection efficiency differences. The HCMV and SCMV β-galexpression vectors were identical to each other in all respects with theexception of the promoter sequences.

RD cells were transfected with the HCMV β-gal reporter plasmid, the SCMVβ-gal reporter plasmid, or a mixture of the two plasmids (H+SCMV). Forsingle plasmid transfections, reporter plasmid DNA amounts were 50-400ng. Total DNA per transfection was held constant at 2.5 μg by adding apromoterless control plasmid. In order to control for transfectionefficiencies and to be able to compare transfection values of differentconstructs at different amounts of DNA in the transfection mixture, thetwo plasmids were mixed in equal proportions and used in thetransfection. For the mixed plasmid transfection (H+SCMV), equal amountsof each plasmid were used (e.g., the 50 ng transfection contained 25 ngof each plasmid). In the linear range of transfection, at DNA levelsbelow the K_(m) the plot of initial velocity over DNA added to cells ispredicted to be linear. The slope of the line derived for the mixture ispredicted to be an average of the slopes of the two lines derived forHCMV and SCMV, if transfection efficiencies were similar.

The relative expression level of HCMV to SCMV (2.022) is derived fromthe ratio of the slopes (1.998÷0.988). The differences, however, mayreflect variations in transfection efficiencies rather than truedifferences in relative expression levels. Variations in transfectionefficiency were measured in a mixed transfection.

If no differences in transfection efficiency exist between the HCMV andSCMV preparations, a mixed plasmid transfection will yield a theoreticalslope of 1.493. The presence of inhibitors or enhancers of transfectionin one of the preparations would alter the theoretical slope. In fact,the mixed transfection generated a lesser slope (1.195) than thetheoretical slope, indicating that the reduced expression of SCMV was,in part, a result of differences in transfection efficiency. The levelof repression caused by transfection inhibition (caused by elements oftranscription prior to the entry of DNA into cells) can be calculated bydividing the theoretical slope by the experimentally derived slope ofH+SCMV; 1.255 in this experiment. Expression of SCMV is then correctedby multiplying the experimental value (0.988) by the repression factor(1.255). The true relative activity is then determined by dividing HCMVactivity by the corrected SCMV activity.

Experiment #5

A similar kinetic analysis was performed to study the effects of otherpromoters and other genetic elements. FIG. 6 shows the effect of theenhancer element on the elongation factor alpha promoter (EF-1αpromoter). Publication WO 02/50264, which is incorporated herein byreference, contains the EF-1a promoter (SEQ ID No. 1).

Promoter-Intronless “Enhancer” Sequence. Eƒ-1α Px construct

A promoter sequence was constructed with the following sequences: aminimal EF-1a promoter (Pmin) (nucleotides 1-204 of SEQ ID NO: 1),linked to a sequence containing the 5′ exon of the native enhancer(nucleotides 205-238 of SEQ ID NO: 1), which is linked to a sequencecontaining the 3′ exon of the native “enhancer” (nucleotides 1160-1170of SEQ ID NO: 1) but lacks the intron sequence of the native “enhancer”located between the two exons. This synthetic sequence containsnucleotides 1-238 immediately fused to nucleotides 1160-1170 of SEQ IDNO: 1. This construct is referred to as the Ef-1a Px construct. Whenthis sequence was employed as the promoter in the above-describedplasmid construct, it provided a 2-fold increase in expression relativeto the minimal promoter plasmid in expression assays. The expressionwas, however, lower than that obtained using the plasmid constructcontaining the entire native EF-1a promoter-“enhancer” which is referredto herein as the EF-1a PE construct. The inverse plot demonstrates thatthe enhancer element reduces the commitment of the promoter totranscription (increases K_(m)), but increases the maximal rate at whichthe gene is transcribed (increases V_(max)). A schematic diagram of theEF-1a promoter organization and the constructs used in the studiesdescribed herein are shown in FIG. 17.

Another experiment (FIG. 7) compares the kinetics of the HCMV promoterwith the EF-1α Pmin promoter construct. The inverse plot demonstratesthat the HCMV promoter has a relatively lower commitment totranscription. Therefore, the HCMV promoter would not be expected to beactive at a low copy number and is not predicted to be useful undersub-saturating transfection (DNA uptake) conditions. However, the EF-1αPmin is predicted to be efficient in achieving commitment at lowtransfection efficiencies (plasmid DNA uptake). The HCMV promoterdemonstrates a very high V_(max)—greater than 30 fold over EF-1α Pmin.To achieve high level expression at very low DNA uptake as would beneeded for gene therapy, DNA vaccine, and RNAi applications, it would beuseful to replace the sequence element of HCMV that contributes to thehigh K_(m) with the EF-1alpha Pmin sequences, as described in Example 2below.

FIGS. 9A and 9B compare the kinetics of the HCMV promoter to the RousSarcoma virus promoter (RSV). Both promoters are approximately equallycommitted to transcription (K_(m)); however, the HCMV promoter resultsin higher expression levels at any concentration of transfection DNA(V_(max)). The experiments shown in FIGS. 6A, 6B, 7A, 7B, 9A, and 9Bwere controlled for differences in transfection efficiency. In allcases, the mixture of the two plasmids resulted in β-gal activityintermediate between the activity observed with each vector alone.Therefore, no DNA uptake efficiency correction was necessary.

Mapping Promoter Elements Using a Genetic Approach

The sequence elements that contribute to K_(m) and V_(max) effects canbe mapped by random mutation either in vitro (e.g., by PCR basedmutagenesis, sequence shuffling, or mutagenizing), in vivo in bacteriaor eukaryotic cells with single or multiple copy plasmids, or usingintegrated sequences (e.g., retroviruses, random integration, Cre-Lox).Once identified, the sequence elements can be spliced together to getthe desired promoter effect, such as a promoter with a low K_(m) and ahigh V_(max).

This kinetic testing methodology is not limited to promoter assessment.These techniques can be used to measure the effects of anytranscriptional or translational control elements, promoters being onlyone example. These methods are also applicable for the optimization oftransfection conditions.

EXAMPLE 2 Construction and Function of a Chimeric HCMV/EF-1α minPromoter (Chimeric HCMV/EF-1α Pmin Promoter)

Generation of Constructs

The minimal promoter of the human elongation factor-1alphapromoter-enhancer (EF-1α Pmin, WO 02/50264) is used to create a chimericpromoter by joining this EF-1α minimal promoter to sequences in the HCMVpromoter. The sequence of the EF-1α minimal promoter, nucleotides 1-204,is as follows:

-   cgtgaggctc cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt    tggggggagg ggtcggcaat tgaaccggtg cctagagaag gtggcgcggg gtaaactggg    aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa    gtgcagtagt cgccgtgaac gttc (SEQ ID NO: 2).

In this particular example, the 3′ terminal 71 base-pairs containing theTATA and CAT boxes are first deleted from the HCMV immediate earlypromoter (GenBank accession # X 03922). The EF-1 αmin promoter elementreplaces these deleted HCMV sequences as depicted in FIG. 8. Cloning ofthe EF-1 αPmin promoter element is described in W002/50264. For cloningthe HCMV promoter containing the 71 base-pair deletion, the followingstrategy is employed. The 5′ portion of the human cytomegalovirus (HCMV)immediate early promoter (nucleotides 370-1070 of Genbank accession no.X03922, which lacks the 3′ terminal 71 base-pairs) is amplified by PCRusing the following oligonucleotide primers: “Forward”5′-TGGCACATGGCCAATGCATT-3′ (SEQ ID NO: 3) and “Reverse”5′-GGCGGAGTTGTTACGACATTT-3′ (SEQ ID NO: 4). The plasmid pCDNA4(Invitrogen, Carlsbad, Calif.) can be used as a template for PCR, sinceit contains the HCMV immediate early promoter. The amplified productdoes not include the CAT and TATA boxes that are important fortranscriptional activity.

The two PCR products are ligated together using T4 DNA ligase and usingstandard techniques. The ligation product is then PCR-amplified usingroutine methods known to those skilled in the art. The ligation productis then cloned into a plasmid vector that does not contain a eukaryoticpromoter element, but contains a reporter gene and a polyadenylationsignal. An example of such a vector is pGL3 (Promega, Madison Wis.).This vector contains a luciferase reporter gene followed by apolyadenylation signal. Different promoters and promoter enhancercombinations can be cloned into this vector upstream of the reportergene. These vectors are then transfected into suitable cells andassessed for luciferase expression.

Vector A contains the Chimeric HCMV/EF-1α Pmin promoter depicted in FIG.20. Vector B contains the full-length wild type HCMV promoter. Thispromoter is generated by PCR using pCDNA 4 as a template and the forwardprimer already described above. The reverse PCR primer for creating thisPCR product maps to the extreme 3′ end of the HCMV immediate earlypromoter. The sequence is: 5′-CGGTTCACTAAACGAGCTCTG-3′ (SEQ ID NO:5).This sequence corresponds to nucleotides 1121-1141 of Genbank accessionno. X03922. The above primer is the reverse complement of that sequence.Vector C contains the EF-1α Pmin promoter. Following PCR amplification,the PCR products are cloned into pGL3 using standard techniques.Analysis of ConstructsHuman rhabdomyosarcoma cells (RD) are seeded into six-well plates. Whencells are 80-95% confluent, they are transfected with varying amounts ofthe experimental constructs and Lipofectamine (Gibco-BRL) according tothe manufacturer's instructions. Total DNA is held constant at 2.5 μgusing pGL3 basic as filler. It is noted that pGL3 basic has no promoterand has been demonstrated not to express detectable levels of luciferasefollowing transfection into RD cells. At 24 and 48 hourspost-transfection, cells are lysed and assayed for luciferase activityusing the Luciferase Assay system (Promega, Madison, Wis.) according tomanufacturer's directions. Luciferase measurements are performed on aluminimeter. An example of a luminometer is the Berthold Microlumat LB96P. V_(max) and K_(m) values are derived following expression analysisas described herein.

Desirably, the chimeric HCMV/EF-1α Pmin promoter has a lower K_(m) thanthe HCMV promoter and a V_(max) that lies between the V_(max) values ofthe EF-1α Pmin promoter and the HCMV promoter. Accordingly, one aspectof the invention provides a nucleic acid molecule encoding the chimericHCMV/EF-1α Pmin promoter. In another aspect, the invention provides anucleic acid molecule in which the chimeric HCMV/EF-1α Pmin promoter isoperably linked to an RNA or protein coding sequence of interest. Alsoenvisioned are methods of expressing an RNA or protein of interestutilizing expression constructs comprising the chimeric HCMV/EF-1α Pminpromoter.

Other chimeric promoters in which the EF-1α Pmin promoter is positionedwithin other regions of the HCMV promoter are expected to exhibitadvantageous characteristics. In addition, other chimeric promoterconstructs incorporating the EF-1 a Pmin promoter and other regulatoryelements can be readily constructed by those of skill in the art andevaluated utilizing the methods disclosed herein. Other promoters inaddition to HCMV that are useful for such manipulations include, but arenot limited to, promoters from Simian Virus 40 (SV40), Mouse MammaryTumor Virus (MMTV) promoter, Human Immunodeficiency Virus (HIV) such asthe HIV Long Terminal Repeat (LTR) promoter, Moloney virus, ALV,Cytomegalovirus (e.g., HCMV, such as the CMV immediate early promoter,as well as MCMV and SCMV), Epstein Barr Virus (EBV), Rous Sarcoma Virus(RSV) as well as promoters from human genes such as human actin, humanmyosin, human hemoglobin, human muscle creatine, human metalothionein,and human mitochondrial promoter

The preparation or synthesis of the nucleotide sequences and cloningtechniques described herein are well within the ability of a personhaving ordinary skill in the art using available material.

EXAMPLE 3 Forced Open Promoter Complex

The function of a promoter is to affect transcriptional initiation, therate-limiting step in transcription. The traditional approach toimproving promoter functionality has been through the identification andoptimization of nucleic acid sequences with desirable properties whichresult in high level transcription initiation. To date, attempts tocreate an unregulated promoter have been unsuccessful.

The rate of transcription initiation is limited by open complexformation (melting of the transcriptional start site), and is dependenton the promoter sequence and the transcription factors that bind thepromoter sequences (FIG. 9). A bubble of unbase-paired DNA resembles thetranscription bubble associated with open promoter complexes.Transcription from these bubbles is stimulated in excess of ahundred-fold relative to the completely duplexed promoter elements,suggesting that blocking complete base-pairing within a promotersequence can generate open promoter complexes (Tantin and Carey, 1994 J.Biol. Chem. 269, 17397-17400 and Pan and Greenblatt, 1994 J. Biol. Chem.269, 30101-30104). Supercoiled DNA throws out single stranded DNA loopsin order to release torsional stress. Loop formation is random anddynamic (FIG. 10) (Bentin and Nielsen 1996 Biochem. 35, 8863-8869). Whenoligonucleotide concentrations are high relative to plasmidconcentrations, the loops anneal to complementary oligonucleotidesinstead of the partner plasmid strand. This creates a heteroduplex.Furthermore, potassium permanganate probing reveals that the regions ofplasmid DNA on either side of the annealed oligonucleotide are notbase-paired with the partner plasmid DNA strand. This base-pairing ispresumably thermodynamically unfavorable. The heteroduplex between theoligonucleotide and the supercoiled vector, creating a free singlestrand in the vector, creates an open promoter complex which favorstranscriptional initiation.

An unregulated, or super promoter, is formed if the heteroduplex isstabilized for an indefinite period of time. We have discovered thatstabilization is enhanced by creating a heteroduplex “padlock” on thepromoter sequence. DNA padlocking is performed by incubating supercoiledplasmids with a relatively high concentration (e.g.,1000 fold molarexcess) of a linear DNA (padlock) containing a 5′ end that iscomplementary to a sequence of the target DNA molecule, and a 3′ endthat is complementary to a contiguous sequence of the target DNAmolecule. The linear DNA molecule base-pairs to the target supercoiledplasmid, thereby juxtaposing the 3′ and the 5′ ends of the linearmolecule annealed to adjacent bases on the target sequence. The regionsof complementarity of the 5′ and 3′ ends of the DNA must be contiguousand should each be at least five nucleotides in length. Once inposition, the two ends of the linear sequence are ligated to form acircular nucleic acid (e.g., DNA) with at least 10 bases (at least fiveat each end of the linear sequence that are annealed to the target) thatare hydrogen-bonded to the target sequence. This ligation allows theoligo to contain at least one linking number with respect to the targetsequence resulting in one helical turn. In various embodiments, theoligonucleotide is a DNA, an RNA, a PNA, or a mixture thereof. Indesirable embodiments, the single stranded oligonucleotide comprises atleast five contiguous nucleotides at the 5′ terminus that arecomplementary to a first region of the supercoiled DNA or RNA and atleast five contiguous nucleotides at the 3′ terminus that arecomplementary to a second region of the supercoiled DNA or RNA that isadjacent to the first region. Desirably, the first and/or second regionsof one strand of the supercoiled DNA or RNA that are 100% complementaryto the 5′ or 3′ terminus of the single stranded oligonucleotide are atleast 6 to 10 or 10 to 20 nucleotides, more desirably at least 30nucleotides, most desirably at least 40 nucleotides or even at least 50nucleotides or 100 nucleotides in length. The first and second regionsof complementarity may be the same or different lengths. The schematicdiagram in FIG. 18 depicts the design features of forced open complexesprepared using an oligo or a padlock with respect to the transcriptionstart site. The 3′ and 5′ complementary sequences on the supercoiled DNAor RNA are next to each other so that when the oligonucleotide isannealed to the supercoiled DNA or RNA, the 3′ and the 5′ terminalnucleotides of the single stranded oligonucleotide base-pair withadjacent bases in the supercoiled DNA or RNA and can be ligated 3′ to 5′to form a molecule torsionally locked to the supercoiled DNA or RNA. Inone aspect, a phosphodiester linkage is formed between the 3′ and the 5′ends of the single stranded oligonucleotide. In addition to thecomplementary 5′ and 3′ sequences, the oligonucleotide desirablyincludes a region linking the 5′ and the 3′ ends that is at least aslong, and desirably longer to avoid torsional stress, than the totallength of the complementary 5′ and the 3′ sequences. In one embodiment,the linking region consists of nucleotides with naturally-occurring ormodified bases.

The intervening, connector sequence (linking region) can be a randomsequence that is not complementary to any portion of the vector, or canbe a sequence useful for targeting a cell type, or a sequence that bindseither a polymerase or transcription factor and desirably enhancestranscription by bringing this factor or polymerase to proximity withrespect to the padlocked sequence. Examples of such useful, non-randomsequences include sequences that promote RNA polymerase binding, such asminimal sequences of EF-1alpha. Subsequent to annealing of the vectorDNA and the padlock DNA, the padlock DNA is ligated by chemical orenzymatic means (FIG. 11). Chemical ligation can be carried out usinglinking technologies such as those using maleimide, carbodiimide, orsuccinamide technologies (Pierce), or the hydrazine-aldehyde linkagesdescribed by Solulink (San Diego, Calif.). For example, anoligonucleotide with an aldehyde can be ligated to an oligonucleotidewith a hydrazine on a DNA template, provided the two oligonucleotidesare in proximity due to base-pairing to the template such that the endbases of the oligonucleotides become adjacent. Chemical linkage may alsobe accomplished as described in Luebke and Dervan, J. Am. Chem. Soc.,1989, 111: 8733-8735 and Luebke and Dervan, JACS, 1991, 113. Exemplarytemplate-directed chemical ligation methods are described by Herrleinand Letsinger, Nucleic Acids Res. 1994 Nov 25:22(23):5076-8; Gryaznovand Letsinger, Nucleic Acids Res. 1993 Mar 25;21(6):1403-8; Calderone etal., Angew. Chem. Int. Ed. 2002, 41, No. 21, page 4104; Gartner et al.,J. Am. Chem. Soc. (JACS) 2002, 124, 10304-10306; Gartner et al., Angew.Chem. Int. Ed. 2002, 41, No. 10, page 1796; Gartner and Liu, J. Am.Chem. Soc. 2001, 123, 6961-6963. Ligation of the 5′ and 3′ ends of thepadlock DNA forms a knotted structure, where the padlock DNA is wrappedaround the vector DNA once for about every ten nucleotides. Theresulting heteroduplex is, therefore, stabilized indefinitely, forming apermanent forced-open complex and should be unaffected bytranscriptional repressors, or a lack of transcriptional enhancers.

According to this invention, this technique is not limited to use ofpreviously identified promoter sequences. A permanent forced-opencomplex can be formed at any DNA sequence and acts to promotetranscription of the downstream sequence. The schematic diagram in FIG.19 depicts the design features of forced open complexes prepared usingan oligo or a padlock with respect to the translation initiation site.

EXAMPLE 4 Forced Open Promoter Complexes

Four oligonucleotides (46-mers) are synthesized. SC1001, SC1002, andSC1003 are each DNAs complementary to a region of the HCMV promoter(FIG. 20), whereas SC1004 is a scrambled oligo. SC1001 is a 46-mer thatoverlaps the CAT and TATA boxes of the HCMV promoter as contained in theplasmid pCMV-beta (BD Biosciences, Palo Alto, Calif.; GenBank accession#: U02451). SC1002 is a 46-mer that extends to the −1 site of the HCMVpromoter. SC1003 is a 46-mer that straddles the +1 site of the HCMVpromoter. SC1004 is a scrambled 46-mer with the same nucleotidecomposition as SC1001, but without complementarity to any region ofpCMV-beta. Each of the oligonucleotides is incubated with pCMV-betavector DNA at a 1000-fold molar excess under standard conditionsdesigned to favor annealing. Various amounts of pCMV-beta complexed witholigonucleotides, including 0.2, 0.4, 0.6, 0.8 or 1 ng of the vector,are used to titrate the range of expression. Each of the annealedoligonucleotide-vector complexes is transfected into 8.5×10⁵Rhabdomyosarcoma (RD) cells using Lipofectamine (Invitrogen, Carlsbad,Calif.) according to the manufacturer's instructions. As controls, cellsare transfected with various amounts of pCMV-beta alone, including 0.2,0.4, 0.6, 0.8 or 1 ng of the vector per well. The total amount of DNA inall the transfections is adjusted to 2.5 μg with pGL3 (Promega, Madison,Wis.), which acts as filler DNA. At 48 hours post-transfection, thecells are lysed with 500 ul of lysis buffer (300 mM NaCl; 50 mM Tris, pH7.6; 0.5% Triton X-100; and 0.5% Sodium deoxycholate) for one hour at 4°C. with rocking. Promoter activity is monitored by assaying forβ-galactosidase activity according to the protocol described in Sambrooket al. (1989), modified as described in Examples 5A-5C below. K_(m) andV_(max) values are calculated by procedures described herein.

Desirably, the complementary oligonucleotides yield annealed promotercomplexes which function as strong promoters with very low K_(m) values.Desirably, increases of 50 to 500 fold in V_(max) are observed,depending upon the oligonucleotide selected. The forced open promotercomplexes desirably have K_(m) values at least several hundred foldlower than the native HCMV promoter, indicating that theseoligo-annealed promoter constructs may be active at only a few copies oreven a single copy per cell. The SC1001-HCMV construct is expected toshow maximum enhancement of the reporter expression compared to thecorresponding values from cells transfected with uncomplexed reporterplasmid, pCMV-beta. The SC1004 oligonucleotide (which is notcomplementary to any region of pCMV-beta and does not hybridize to it)is expected to show negligible enhancement over the reporter plasmidalone. The following exemplary sequences may be used:

-   SC1001 (inverse complement of nucleotides 484-529 of pCMV-beta;-   5′-tgcttatatagacctcccaccgtacacgcctaccgcccatttgcgt-3′(SEQ ID NO: 6),    SC1002 (inverse complement of nucleotides 503-548 of pCMV-beta;-   5′-cggttcactaaacgagctctgcttatatagacctcccaccgtacac-3′(SEQ ID NO: 7),    SC1003 (inverse complement of nucleotides 526-571 of pCMV-beta;-   5′-gatggcgtctccaggcgatctgacggttcactaaacgagctctgct-3′(SEQ ID NO 8),    and SC1004 (scrambled version of SC1001;-   5′-gatgatcggatcgagtcggagatcgatggatcggatcggatcgagtcgagtcag-3(SEQ ID    NO: 9).

EXAMPLE 5A Generation of Padlocked Supercoiled DNA

The HCMV-β-gal supercoiled plasmid (construct 017) and the pShooterplasmid (Invitrogen) were heated to 95° C. to produce a mixed solutionof linear, nicked, and supercoiled plasmid DNA. Plasmid DNA wasincubated with linear padlock DNA (³²P-labeled), with the 5′ and 3′ endsbeing complementary to a continuous 38 nucleotide sequence covering theCAT and TATA box of the HCMV promoter. After annealing, the padlock DNAwas ligated using a thermostable ligase (Epicenter Inc., Wisconsin,U.S.A.) and separated on an agarose gel. FIG. 15 is a photograph of theagarose gel stained with ethidium bromide, showing the presence of allthree forms of DNA (supercoiled, nicked, and linear). Also included isan autoradiogram of the same gel showing that only supercoiled DNA iscapable of being padlocked. Additionally, the padlock did not bind tothe control plasmid.

EXAMPLE 5B Forcing Open known Promoter Sequences with Padlocks

The single stranded oligonucleotide sequence or the torsionally lockedpadlock oligonucleotide is designed to be complementary to the sequencesof the promoter. The ideal method to enhance promoter expression is todesign the base-pairing to extend into the promoter region, desirablybeginning at sequences −1 to the transcription initiation site on thenon-coding strand (i.e, the non-template or non-transcribed strand) ofDNA. Alternatively, the oligonucleotide may be designed such that thebase-pairing may begin further into the promoter, e.g., at the −45, atthe −20, or at the −10 sequence position. Base-pairing to the codingstrand (i.e., the transcribed strand) is less desirable. Thebase-pairing window (i.e., length of complementary sequence) shouldminimally be 10-12 nucleotides, more desirably 20 to 30, and mostdesirably 45 nucleotides, but can be up to 100 nucleotides in length.Many RNA polII promoters contain either or both the TATA box at about−25 and the CAT box at or near the −45 sequence region preceding thetranscription initiation site. Base-pairing to one or desirably bothelements (e.g., the TATA and CAT box) may be used in some embodiments.Desirably, the oligonucleotide base-pairs to the element nearest to the−1 position.

While a complementary torsionally locked padlock oligonucleotide, DNA,RNA, or PNA provides a stable, forced open promoter that is particularlyadvantageous for certain applications, it is recognized thatnon-padlocked linear or circular single stranded olignucleotidesequences, (e.g., DNA, RNA, PNA, or hybrids thereof) may also beannealed as described to promoter sequences to provide forced openpromoters with enhanced, though less stable, activity. Single strandedor circular non-padlocked oligonucleotides, (e.g., DNA, RNA, PNA, orhybrids therof) are annealed as described herein to one strand of adouble stranded DNA or RNA (desirably to a promoter sequence or anothersequence upstream from and operably linked to an RNA or protein codingsequence of interest) but not torsionally locked between the two DNA orRNA strands as with a padlock oligonucleotide. Furthermore, while suchforced open promoters are advantageously prepared using promoters foundin supercoiled DNA or RNA expression constructs or vectors, thesemethods and constructs are also applicable to forced open promoterswithin nicked, covalently closed circular, or linear DNA or RNAexpression constructs, as well as other non-promoter sequences withinsuch supercoiled, nicked, closed circular, or linear expressionconstructs capable of expressing mRNA and optionally also protein. Whileit is desirable to make the various forced open promoter constructs onsupercoiled DNA or RNA, if the construct is subsequently nicked,linearized, or converted to a covalently closed circular DNA or RNA, itwill still function as described herein, albeit with potentially alteredefficiency.

EXAMPLE 5C Generation of a Padlocked HCMV Promoter

Five ng pCMV-beta plasmid (BD Biosciences, Palo Alto, Calif.; GenBankaccession #:U0245 1) is incubated with 1000-fold molar excess of padlockoligonucleotide #1 (described below) under conditions favoring annealingof padlock oligo to pCMV-beta. The sequence of the padlock oligo isdesigned to anneal to the HCMV promoter in the plasmid. Incubation iscarried out in a volume of 15 ul of AmpLigase buffer and 5 u AmpligaseThermostable DNA ligase (Epicentre, Madison, Wis.) in a thermocycler.Incubation temperature is initially set at 65° C. The temperature isramped down by 2° C. every 10 minutes until a final temperature of 50°C. is reached, at which point the reaction is terminated. During thereaction, the 3′ and the 5′ ends of the oligo are annealed to contiguoussequences of pCMV-beta. Then, ligation of the abutting 3′ and 5′ ends ofthe annealed oligo occurs, generating the padlocked oligo structureshown in FIG. 16. As shown, padlock oligonucleotide #1 is annealed topCMV-beta. The 5′ twenty-eight nucleotides base-pair with nucleotidesmapping to coordinates −47 to −20 of the HCMV promoter present inpCMV-beta. The internal nucleotides do not base-pair with pCMV-beta.They are included to (i) give the oligonucleotide freedom to rotate itstermini to facilitate base-pairing and (ii) prevent additional torsionalstress to the supercoiled pCMV-beta following annealing of the padlockoligo. Once annealed, the oligo is ligated to create a phosphodiesterlinkage between the 3′ and the 5′ termini of the oligo generating thepadlocked oligo structure. Regions flanking the annealed padlock oligoare single-stranded due to torsional constraints.

One advantage of using padlocked oligonucleotides relative to annealedsingle-stranded non-padlocked oligonucleotides is that padlockedoligonucleotides are stably annealed to the DNA and are only releasedfollowing nicking of the oligo or the region of DNA to which the oligois annealed. This factor makes plasmids containing padlockedoligonucleotides ideal for in vivo use, although they can be used for invitro applications as well. For certain applications, linearoligonucleotides annealed to promoter sequences in supercoiled DNA orRNA plasmids can advantageously be employed to achieve enhancedexpression similar to that described below, although less sustained forin vivo applications. In some applications this more transient effect isadvantageous.

The Padlock oligo #1 sequence is presented below:

5′GTGTACGGTGGGAGGTCTATATAAGCAGTCGAGTTAATTAACGGCCGTCTAGAGGTACCGAATTCGCTAGCGCGGCCGCCGATCGGTCGACGGACGCAAATGGGCGGTAGGC3′(SEQ ID NO: 10). Underlined sequences represent those oligo sequencesthat bind to sequences in the HCMV promoter as depicted in FIG. 16. The5′underlined sequences (5′-3′ direction) map to coordinates 503-530 ofGenBank accession number U02451, and the 3′ underlined sequences (5′-3′direction) map to coordinates 483-502 of GenBank accession numberU02451. These sequences represent coordinates −47 to −20 and −67 to −48of the HCMV promoter, respectively.Transfection

The annealing and ligation reaction mix is sufficient for fivetransfections. Transfection mixtures are comprised of 3 ulannealing/ligation mixture (corresponding to about 1 ng padlockedpromoter plasmid) and 2.5 μg pGL3 basic (Promega, Madison, Wis.) asfiller DNA in a volume of 10 ul. Approximately 8.5×10⁵ RD cells aretransfected using Lipofectamine™ (Invitrogen, Carlsbad, Calif.)according to the manufacturer's directions. Control cells aretransfected with transfection mixtures containing 1 ng, 5 ng, 50 ng, or100 ng unpadlocked pCMV-beta. These transfection mixtures contain pGL3basic DNA as filler DNA such that the total amount of DNA pertransfection is 2.5 μg. At 48 hrs post-transfection, the cells are lysedwith 500 ul lysis buffer (300 mM NaCl: 50 mM Tris, pH 7.6, 0.5% TritonX-100, and 0.5% NaDeoxycholate) for one hour at 4° C., with rocking.Promoter activity is monitored by assaying for B-galactosidase using amodification of the protocol described in Molecular Cloning, ALaboratory Manual, 2 nd Edition, Sambrook et al., Cold Spring HarborPress, Plainview, N.Y. (1989). The modification is that readings aretaken every minute for 90 minutes following setting up the B-Gal enzymeassay. Readings are taken on a kinetic plate reader such as the VersMaxTunable Microplate reader (Molecular Devices, Sunnyvale, Calif.).V_(max) values are calculated by procedures determined herein.Desirably, the padlocked pCMV-beta plasmids generate higher V_(max)values following transfection relative to the unpadlocked pCMV-betaplasmids. In desirable embodiments, values are typically 50-500 foldhigher, which would indicate that 50-500 fold less plasmid can beadministered (relative to unpadlocked plasmid) without a loss inexpression.

EXAMPLE 5D Generation of Padlocked Promoter

The expression vector used for these experiments is pCMV-SEAP, aproprietary eukaryotic SEAP (placental heat-stable SEcreted AlkalinePhosphatase) expression vector. This vector contains the SEAP expressioncassette from pSEAP2-Control plasmid (Clontech, Palo Alto, Calif.;GenBank Accession# U89938) and includes the SEAP coding region and the5′UTR (coordinates 249-1831 of pSEAP2-Control). This region was clonedinto a plasmid vector under the transcriptional control of the HCMVpromoter described herein. A BGH polyadenlyation site provides thepolyadenylation signal. Cloning is performed using standard techniques.Approximately 100 ng pCMV-SEAP is incubated with 1000-fold molar excessof padlock oligonucleotide #1 described above under conditions favoringannealing of padlock oligo to pCMV-SEAP. The reaction is split intothree reaction tubes of 100 ul each. The reaction mixture includes 1×Ampligase buffer, and each 100 ul reaction contains 35 u of Ampligase(Epicentre, Madison, Wis.). The padlock oligo is designed to anneal tothe HCMV promoter. The incubation is carried in a thermocycler and theincubation temperature is initially set at 65° C. The temperature isramped down by 2° C. every 10 minutes until a final temperature of 50°C. is reached at which point the reaction is terminated. During thereaction, annealing of the oligo to pCMV-SEAP occurs. Then, ligation ofthe 5′ and 3′ ends of the annealed oligo occurs, generating a padlockedoligo structure. One advantage of using padlocked oligonucleotidesrelative to annealed linear oligonucleotides is that padlockedoligonucleotides are stably annealed to the DNA and are only releasedfollowing nicking of the oligo or the region of DNA to which the oligois annealed.

To test the in vivo effects of a padlock, the padlocked pCMV-SEAPplasmid constructs and unpadlocked pCMV-SEAP plasmids are prepared in 30mM citrate buffer (0.1 5M NaCl, 0.1% EDTA and 0.25% bupivicaine), pH 6.5to 6.8, at a concentration of 0.1 mg/ml of the padlocked pCMV-SEAPplasmid. Inert filler DNA, pGL3 basic, described herein, is included tobring total DNA concentration to 2 mg/ml. The unpadlocked DNA constructis prepared similarly except that in addition to being formulated at 0.1mg/ml, additional formulations of 0.50 mg/ml, 1 mg/ml and 2 mg/ml areprepared. Final DNA concentrations are all adjusted to 2 mg/ml with pGL3basic. A 100 ul dose of each formulation is injected intramuscularlyinto the quadriceps of mice. There are five mice per each formulationgroup. At five days postinjection when in vivo plasmid expression peaks,blood is taken from the injected mice by retro-orbital bleed, and thelevel of SEAP in the sera is determined using the assay described below.

SEAP activity is determined in a kinetic SEAP enzyme assay. According tothe SEAP assay, 5-10 ul heat-inactivated sera is added to each well in a96-well plate. Substrate solution (200 ul) prepared by dissolving 1 vialALP 20 (alkaline phosphatase, Sigma 245-20) in 20 mL water at roomtemperature is added to each well. Each well is read immediately usingan OPTI max tunable microplate reader (Molecular Devices Co., Sunnyvale,Calif.) at 405 nm kinetic mode, and additional readings are taken everyminute for 60 minutes. The linear window of the plot is used foranalysis. The slopes of the linear plot are used to compare the relativein vivo expression levels.

Mice injected with the formulation containing the padlocked pCMV-SEAPplasmid desirably express SEAP at levels exceeding those levelsoccurring in mice injected with any of the formulations containing thenon-padlocked pCMV-SEAP, which would indicate that expression from anexpression vector can be increased in vivo through the use of padlockoligo invasion of the promoter used to drive expression of the gene ofinterest. Other animal models may be used similarly.

Generating Open Promoter Complexes De Novo

The design of the complementary linear or padlock oligonucleotide issimilar to the one described above, with base-pairing to occur desirablyon the non-coding strand. However, since there is no promoter per se,the −1 position is arbitrarily assigned. The −1 position is selectedsuch that the base-pairing window is at least 45 nucleotides (e.g.,between 80 and 150 nucleotides) prior to the initiating ATG codon. Thesequences between the arbitrary −1 site and the downstream ATG codondesirably conform to all or most of the Kozak rules (e.g., no ATGs, fewor no major secondary structures (DeltaG<40 Kcal)). The design featuresthat relate to positioning the oligo or the padlock to achievetranscription and/or translation are shown in FIG. 19.

The complementary single stranded oligonucleotides and the padlockoligonucleotides described herein may be DNA, RNA, PNA (peptide nucleicacid), or a hybrid thereof (Gambari, Curr Pharm Des 2001Nov;7(17):1839-62). They may also contain backbone modifications. Forexample, inter-nucleotide linkages other than phosphodiester bonds, suchas phosphorothioate, methylphosphonate, methylphosphodiester,phosphorodithioate, phosphoramidate, phosphotriester, or phosphate esterlinkages (Uhlman et al., Chem. Rev. 90(4):544-584, 1990; AnticancerResearch 10: 1169, 1990) may be present in the oligonucleotides toincrease their stability. Oligonucleotide stability may also beincreased by incorporating 3′-deoxythymidine or 2′-substitutednucleotides (e.g., alkyl substitutions) into the oligonucleotides duringsynthesis by providing the oligonucleotides as phenylisourea derivativesor by having other molecules, such as aminoacridine or poly-lysine,linked to the 3′ ends of the oligonucleotides (see, e.g., AnticancerResearch 10:1169-1182, 1990). Modifications of the RNA and/or DNAnucleotides comprising the oligonucleotides of the invention may bepresent throughout the oligonucleotide, or in selected regions of theoligonucleotide (e.g., the 5′ and/or 3′ termini). The oligonucleotidesmay also be modified in their non-base-pairing regions to increase theirability to penetrate a target tissue. For example, the oligonucleotidemay be covalently coupled to lipophilic compounds, receptor ligands, ormoieties known to enhance endosomal escape. The oligonucleotides of theinvention can be made by any method known in the art, including standardchemical synthesis, ligation of constituent oligonucleotides, andtranscription of DNA encoding the oligonucleotides.

The non-base-pairing regions of the single stranded oligonucleotide orpadlock molecule may be modified to contain covalently linked deliveryagents and/or single stranded and double stranded DNA sequences thatspecifically bind transcription factors and polymerases. These regionsmay also contain secondary structure and include known binding sites fortranscriptional factors, e.g., RNA po1II, as well as other polymerasessuch as RNA polII, mitochondrial RNA polymerase, T7, and vaccinia. Whilesingle-stranded, non-padlock oligonucleotides of the invention arefrequently linear molecules, it is contemplated that circularoligonucleotides may also be utilized advantageously. Such circularsingle stranded oligonucleotides may include a region of at least 10-12nucleotides complementary to a target region of a supercoiled DNA or RNAand the non-base-pairing linker region may advantageously include any ofthe modifications described above (e.g., nucleotides with binding sites,receptor ligands to target cells, or agents to promote delivery orendosomal escape).

The non-base-pairing region may include, for example, nucleotides, PEGs,peptides, and/or proteins. Examples of such delivery agents and othercovalent linkages include compositions containing optionalpolynucleotide facilitating agents or “co-agents,” such as a localanaesthetic, a peptide, a lipid (e.g., a cationic lipid), a liposome orlipidic particle, a polycation such as polylysine, a branched, threedimensional polycation such as a dendrimer, a carbohydrate, a cationicamphiphile, a detergent, a benzyl ammonium surfactant, or anothercompound that facilitates polynucleotide transfer to cells.Non-exclusive examples of such facilitating agents or co-agents usefulin this invention are described in U.S. Pat. Nos. 5,593,972; 5,703,055;5,739,118; 5,837,533; International Patent Application No. W096/10038,published Apr. 4, 1996; and International Patent Application NoW094/16737, published Aug. 8, 1994, which are each incorporated hereinby reference. The linkage can be produced using phosphoramiditechemistry methods to link non-nucleosides to the oligonucleotide orpadlock or using standard chemistry methods to modify a nucleotide orbase in the oligonucleotide or padlock. Desirable padlocks andoligonucleotides base-pair with the non-template strand of the DNA.Alternatively, padlocks or oligonucleotides that base-pair to thetemplate strand may be used.

Examples of promoters useful for creating open promoter complexesaccording to the present invention include, but are not limited to,promoters from Simian Virus 40 (SV40), Mouse Mammary Tumor Virus (MMTV)promoter, Human Immunodeficiency Virus (HIV) such as the HIV LongTerminal Repeat (LTR) promoter, Moloney virus, ALV, Cytomegalovirus(HCMV, such as the CMV immediate early promoter, as well as MCMV andSCMV), Epstein Barr Virus (EBV), Rous Sarcoma Virus (RSV) as well aspromoters from human genes such as human actin, human myosin, humanhemoglobin, human muscle creatine, human metalothionein, and humanmitochondrial promoter.

OTHER EMBODIMENTS

It will be readily recognized to those of skill in the art of molecularbiology, biotechnology, and recombinant DNA technology that the forcedopen promoters and promoter constructs of the invention may be useful inany application utilizing transcriptional promoters, including withoutlimitation production of RNA or protein molecules in cell culture and invivo applications such as gene therapy, DNA immunization, and genesilencing (e.g. antisense, PTGS, and RNAi). Exemplary PTGS and RNAiapplications are described in U.S. provisional application Serial No.60/419,532, filed Oct. 18, 2002 and European publication number 1229134,filed Jan.31, 2002.

From the foregoing description, it will be apparent that variations andmodifications may be made to the invention described herein to adopt itto various usages and conditions. Such embodiments are also within thescope of the following claims.

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

1. A method for producing a forced-open promoter complex, wherein saidforced-open promoter complex promotes an increase in transcription of anucleic acid sequence of interest, said method comprising: (i) providinga circular supercoiled DNA comprising the sequence of interest, (ii)annealing to said DNA an oligonucleotide having a 5′ end, a 3′ end, andregion linking the 5′ and 3′ ends, the 5′ end being complementary to afirst region of the DNA that is upstream of said sequence of interestrelative to the direction of transcription, and the 3′ end beingcomplementary to a second region of the DNA that is contiguous to saidfirst region; such that the 5′ and 3′ ends are annealed to adjacentbases of the DNA; and (iii) circularizing the 5′ end of saidoligonucleotide to the 3′ end of said oligonucleotide to topologicallylink the oligonucleotide to the DNA, thereby producing a forced-openpromoter complex and promoting transcription of said sequence ofinterest.
 2. The method of claim 1, wherein the circularizing comprisesligating the 5′ end of said oligonucleotide to the 3′ end of saidoligonucleotide to form a circular padlocked oligonucleotide.
 3. Themethod of claim 2, wherein said ligating stabilizes said forced-openpromoter complex.
 4. The method of claim 1, wherein said forced-openpromoter complex is stable.
 5. The method of claim 2, wherein the 5′ endand the 3′ end of said oligonucleotide anneal to a promoter region ofsaid supercoiled DNA.
 6. The method of claim 5, wherein said promoterregion is operably linked to said sequence of interest.
 7. The method ofclaim 5, wherein said promoter region is naturally occurring orartificially inserted.
 8. The method of claim 1, wherein saidsupercoiled DNA is an expression vector.
 9. The method of claim 1,wherein said oligonucleotide comprises at least 10 nuceltoides.
 10. Themethod of claim 1, wherein said oligonucleotide comprises at least 20nucleotides.
 11. The method of claim 1, wherein said oligonucleotidecomprises at least 30 nucleotides.
 12. The method of claim 1, whereinsaid oligonucleotide comprises at least 40 nucleotides.
 13. The methodof claim 1, wherein said oligonucleotide comprises at least 50nucleotides.
 14. The method of claim 1, wherein the linking region doesnot anneal with said supercoiled DNA.
 15. The method of claim 1, whereinthe linking region comprises a sequence that is useful for targeting ortranscriptional purposes.
 16. The method of claim 15, wherein thelinking region comprises a sequence that promotes binding of apolymerase or a transcription factor.
 17. The method of claim 16,wherein said polymerase is an RNA polymerase.
 18. The method of claim 1,wherein said oligonucleotide anneals to a TATA or CAT box that isupstream from the transcription initiation site of said nucleic acidsequence of interest.
 19. The method of claim 1, wherein said 5′ end andsaid 3′ end are each at least 5 neucleotides in length.
 20. The methodof claim 1, wherein each of said 5′ and 3′ ends of said oligonucleotideis 100% complementary to at least five contiguous nucleotides of saidsupercoiled DNA.
 21. The method of claim 20, wherein at least 10contiguous nucleotides of said supercoiled DNA are base-paired to said5′ or 3′ end of said oligonucleotide.
 22. The method of claim 20,wherein at least 10 contiguous nucleotides of said supercoiled DNA arebase-paired to said 5′ and 3′ ends of said oligonucleotide.
 23. Themethod of claim 1, wherein said forced-open promoter complex promotes anincrease in the transcription of an RNA encoded by said nucleic acidsequence of interest, relative to the transcription of said RNA from asupercoiled DNA that lacks a forced-open promoter complex.
 24. Themethod of claim 1, wherein said forced-open promoter complex promotes anincrease in the expression of a protein encoded by said nucleic acidsequence of interest, relative to the expression of said protein from asupercoiled DNA that lacks a forced-open promoter complex.
 25. Themethod of claim 1, wherein said single-stranded oligonucleotidecomprises a DNA molecule.
 26. The method of claim 1, wherein saidnucleic acid sequence of interest encodes an RNA or a protein.
 27. Themethod of claim 1, wherein the annealing of said oligonucleotide to saidsupercoiled DNA promotes an increase in the expression of said nucleicacid sequence of interest, relative to the expression of the nucleicacid sequence of interest in a supercoiled DNA lacking an annealingoligonucleotide.
 28. The method of claim 1, wherein said oligonucleotideis annealed to said supercoiled DNA at least 45 nucleotides upstreamfrom an initiating ATG codon of said nucleic acid sequence of interest.29. The method of claim 1, wherein said oligonucleotide is annealed tothe non-coding strand of said supercoiled DNA.
 30. The method of claim1, wherein said oligonucleotide is annealed to the coding strand of saidsupercoiled DNA.
 31. A method for increasing the transcription of anucleic acid sequence comprising: (i) forming a forced open promotercomplex in a supercoiled DNA comprising said nucleic acid sequence; (ii)transfecting the resulting supercoiled DNA comprising said forced openpromoter complex into a cell; and (iii) culturing said cell, whereinsaid forced open promoter complex promotes increased transcription ofsaid nucleic acid sequence, relative to the transcription of a nucleicacid sequence in a cell transfected with a supercoiled DNA lacking aforced open promoter complex.
 32. The method of claim 31, wherein step(i) comprises annealing a single-stranded oligonucleotide to saidsupercoiled DNA.
 33. The method of claim 32, wherein saidoligonucleotide is circular.
 34. The method of claim 32, wherein saidoligonucleotide is linear.
 35. The method of claim 32, wherein saidsingle-stranded oligoncleotide anneals to said supercoiled DNA upstreamof said nucleic acid sequence.
 36. The method of claim 34, wherein the5′ end of said oligonucleotide anneals to an upstream end of a targetsequence of said supercoiled DNA and the 3′ end of said oligonucleotideanneals to a downstream end of said target sequence, wherein saidupstream and downstream ends of said target sequence comprise acontiguous sequence of said supercoiled DNA.
 37. The method of claim 36,wherein step (i) further comprises ligating the 5′ end of saidoligonucleotide to the 3′ end of said oligonucleotide to form a circularpadlocked oligonucleotide, wherein said circular padlockedoligonucleotide prevents homoduplex formation within said targetsequence of said supercoiled DNA and promotes transcription of saidnucleic acid sequence downstream of said target sequence.
 38. The methodof claim 36, wherein said target sequence is a promoter region of saidsupercoiled DNA.
 39. The method of claim 38, wherein said promoterregion is operably linked to said nucleic acid sequence.
 40. The methodof claim 39, wherein said promoter region is naturally occurring orartificially inserted.
 41. The method of claim 31, wherein saidsupercoiled DNA is an expression vector.
 42. The method of claim 37,wherein said ligating stabilizes said forced open promoter complex. 43.The method of claim 42, wherein said forced open promoter complex isstable for an indefinite period of time.
 44. The method of claim 31,wherein said oligonucleotide comprises at least 10 nucleotides.
 45. Themethod of claim 32, wherein said oligonucleotide comprises at least 20nucleotides.
 46. The method of claim 32, wherein said oligonucleotidecomprises at least 30 nucleotides.
 47. The method of claim 32, whereinsaid oligonucleotide comprises at least 40 nucleotides.
 48. The methodof claim 32, wherein said oligonucleotide comprises at least 50nucleotides.
 49. The method of claim 36, wherein said oligonucleotidefurther comprises an intervening connector sequence that connects said5′ end to said 3′ end.
 50. The method of claim 49, wherein saidintervening connector sequence lacks a region of complementarity withsaid supercoiled DNA.
 51. The method of claim 49, wherein saidintervening connector sequence comprises a sequence that is useful fortargeting or transcriptional purposes.
 52. The method of claim 51,wherein said intervening connector sequence comprises a sequence thatpromotes binding of a polymerase or transcription factor.
 53. The methodof claim 52, wherein said polymerase is an RNA polymerase.
 54. Themethod of claim 36, wherein said target sequence is a TATA or CAT boxthat is upstream from the transcription initiation site of said nucleicacid sequence.
 55. The method of claim 32, wherein said single-strandedoligonucleotide comprises two regions of at least 5 contiguousnucleotides that are complementary to said supercoiled DNA.
 56. Themethod of claim 36, wherein each of said 5′ and 3′ ends of saidoligonucleotide is 100% complementary to at least five contiguousnucleotides of said supercoiled DNA.
 57. The method of claim 36, whereinat least 10 contiguous nucleotides of said supercoiled DNA arebase-paired to said 5′ or 3′ end of said oligonucleotide.
 58. The methodof claim 36, wherein at least 10 contiguous nucleotides of saidsupercoiled DNA are base-paired to said 5′ and 3′ ends of saidoligonucleotide.
 59. The method of claim 31, wherein said forced openpromoter complex promotes an increase in the expression of an RNAencoded by said nucleic acid sequence, relative to the expression of anRNA encoded by a nucleic acid sequence in a supercoiled DNA that lacks aforced open promoter complex.
 60. The method of claim 31, wherein saidforced open promoter complex promotes an increase in the expression of aprotein encoded by said nucleic acid sequence, relative to theexpression of a protein encoded by a nucleic acid sequence in asupercoiled DNA that lacks a forced open promoter complex.
 61. Themethod of claim 32, wherein said single-stranded oligonucleotidecomprises a DNA molecule.
 62. The method of claim 31, wherein said cellis in cell culture, a tissue, or an organism.
 63. The method of claim31, wherein said nucleic acid sequence encodes an RNA or a protein. 64.The method of claim 32, wherein annealing said oligonucleotide to saidsupercoiled DNA promotes an increase in the expression of said nucleicacid sequence, relative to the expression of a nucleic acid sequence ina supercoiled DNA lacking an annealed oligonucleiotide.
 65. The methodof claim 32, wherein said oligonucleotide is annealed to saidsupercoiled DNA at least 45 nucleotides upstream from an initiating ATGcodon of said nucleic acid sequence.
 66. The method of claim 32, whereinsaid oligonucleotide is annealed to the non-coding strand of saidsupercoiled DNA.
 67. The method of claim 32, wherein saidoligonucleotide is annealed to the coding strand of said supercoiledDNA.
 68. A composition comprising: a circular supercoiled DNA containinga sequence for expression; and a padlocked oligonucleotide annealedupstream of said sequence for expression relative to the direction oftranscription, so as to enhance transcription of said sequence forexpression relative to the transcription of said sequence from asupercoiled DNA lacking a padlocked oligonucleotide.
 69. The compositionof claim 68, wherein said padlocked oligonucleotide produces aforced-open promoter complex that prevents homoduplex formation upstreamof said sequence for expression, and thereby promotes transcription ofsaid sequence for expression.
 70. The composition of claim 69, whereinsaid forced-open promoter complex is stable.
 71. The composition ofclaim 68, wherein said supercoiled DNA is an expression vector.
 72. Thecomposition of claim 71, wherein said padlocked oligonucleotide isannealed to a promoter sequence.
 73. The composition of claim 72,wherein said promoter sequence is operably linked to said sequence forexpression.
 74. The composition of claim 72, wherein said promotersequence is naturally occurring or artificially inserted.
 75. Thecomposition of claim 68, wherein said oligonucleotide comprises at least10 nucleotides.
 76. The composition of claim 68, wherein saidoligonucleotide comprises at least 20 nucleotides.
 77. The compositionof claim 68, wherein said oligonucleotide comprises at least 30nucleotides.
 78. The composition of claim 68, wherein saidoligonucleotide comprises at least 40 nucleotides.
 79. The compositionof claim 68, wherein said oligonucleotide comprises at least 50nucleotides.
 80. The composition of claim 68, wherein said padlockedoligonucleotide contains an intervening connector sequence lacking aregion of complementarity with said supercoiled DNA.
 81. The compositionof claim 80, wherein said intervening connector sequence comprises asequence that is useful for targeting or transcriptional purposes. 82.The composition of claim 81, wherein said intervening connector sequencecomprises a sequence that promotes binding to a polymerase ortranscription factor.
 83. The composition of claim 82, wherein saidpolymerase is an RNA polymerase.
 84. The composition of claim 68,wherein the padlocked oligonucleotide is annealed to a TATA or CAT boxthat is upstream from the transcription initiation site of said sequencefor expression.
 85. The composition of claim 68, wherein the annealedregion is at least 10 nucleotides.
 86. The composition of claim 68,wherein said padlocked oligonucleotide comprises a DNA molecule.
 87. Thecomposition of claim 68, wherein said sequence for expression encodes anRNA or a protein.
 88. The composition of claim 68, wherein saidpadlocked oligonucleotide is annealed to said supercoiled DNA at least45 nucleotides upstream from an initiating ATG codon of said sequencefor expression.
 89. The composition of claim 68, wherein said padlockedoligonucleotide is annealed to the non-coding strand of said supercoiledDNA.
 90. The composition of claim 68, wherein said padlockedoligonucleotide is annealed to the coding strand of said supercoiledDNA.
 91. The composition of claim 68, wherein said padlockedoligonucleotide promotes increased transcription initiation at the siteof said sequence for expression.
 92. The composition of claim 72,wherein said promoter sequence is an HCMV promoter sequence.
 93. Acomposition comprising an HCMV promoter annealed to a torsionally lockedoligonucleotide.
 94. A chimeric HCMV/EF-1α Pmin promoter.
 95. A nucleicacid expression construct comprising the chimeric HCMV/EF-1α Pminpromoter of claim 94 operably linked to a nucleic acid sequence.
 96. Thenucleic acid expression construct of claim 94, wherein said nucleic acidsequence encodes an RNA or protein.
 97. A composition comprising asupercoiled DNA comprising a promoter sequence annealed to a torsionallylocked oligonucleotide, wherein said promoter sequence is operablylinked to a nucleic acid sequence.
 98. The composition of claim 97,wherein said promoter sequence is an HCMV promoter sequence.
 99. Thecomposition of claim 97, wherein said nucleic acid sequence encodes anRNA or a protein.
 100. The composition of claim 97, wherein saidoligonucleotide is annealed to the non-coding strand of said supercoiledDNA.
 101. The composition of claim 97, wherein said oligonucleotide isbase-paired with said promoter sequence beginning at sequences −1 to thetranscription initiation site of said nucleic acid sequence.
 102. Thecomposition of claim 97, wherein said oligonucleotide is base-pairedwith said promoter sequence within a region of about −10 to about −70from the transcription initiation site of said nucleic acid sequence.103. A method for generating an open-promoter complex, comprising: (i)providing a purified plasmid in supercoiled form, the plasmid containinga sequence for expression; (ii) creating a padlocked oligonucleotide togenerate an open-promoter complex for said sequence for expression. 104.The method of claim 103, wherein the sequence for expression is operablylinked to a promoter.
 105. The method of claim 104, wherein thepadlocked oligonucleotide is annealed to a TATA or CAT box within saidpromoter.
 106. The method of claim 104, wherein said padlockedoligonucleotide is annealed to the non-coding strand of the supercoiledDNA.
 107. The method of claim 104, wherein said padlockedoligonucleotide is annealed to the coding strand of the supercoiled DNA.108. The method of claim 103, wherein the sequence for expression is notoperably linked to a promoter.
 109. The method of claim 108, wherein thesequence for expression contains an initiating ATG codon or is a dsRNAcoding region.
 110. The method of claim 109, wherein said padlockedoligonucleotide is annealed to the non-coding strand of said supercoiledDNA.
 111. The method of claim 109, wherein said padlockedoligonucleotide is annealed to the coding strand of said supercoiledDNA.
 112. A composition, comprising: a purified plasmid DNA insupercoiled form, the construct containing a sequence for expression;and a padlocked oligonucleotide annealed to said construct, wherein thepadlocked oligonucleotide generates an open-promoter complex for saidsequence for expression.
 113. The composition of claim 112, wherein thesequence for expression is operably linked to a promoter.
 114. Thecomposition of claim 113, wherein the padlocked oligonucleotide isannealed to a TATA or CAT box within said promoter.
 115. The compositionof claim 113, wherein said padlocked oligonucleotide is annealed to thenon-coding strand of the supercoiled DNA.
 116. The composition of claim113, wherein said padlocked oligonucleotide is annealed to the codingstrand of the supercoiled DNA.
 117. The composition of claim 112,wherein the sequence for expression is not operably linked to apromoter.
 118. The composition of claim 117, wherein the sequence forexpression contains an initiating ATG codon or is a dsRNA coding region.119. The composition of claim 118, wherein said padlockedoligonucleotide is annealed to the non-coding strand of the supercoiledDNA.
 120. The composition of claim 118, wherein said padlockedoligonucleotide is annealed to the coding strand of the supercoiled DNA.121. The composition of claim 97, wherein said oligonucleotide isannealed to the coding strand of said supercoiled DNA.